The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe phenotype, non-alcoholic steatohepatitis (NASH), are increasing significantly in the United States in the adult and pediatric population. NAFLD is associated with obesity and metabolic syndrome and its prevalence has increased in parallel to the prevalence of obesity and type-2 diabetes. The development of NAFLD, its different phenotypes, and the heterogeneity of disease progression are not completely understood. Recent evidence suggests that there is an association between intestinal microbial colonization (the intestinal microbiome) and obesity in humans and in animal models. In addition, there is evidence of abnormalities of bacterial colonization, and intestinal bacterial product induced inflammation associated with NAFLD and progression to NASH. The goal of this proposal is to investigate the composition of the intestinal microbiome in pediatric patients with obesity and obesity plus NAFLD, and determine the relationship between alterations in the intestinal microbiome, immune activation, and the development of NAFLD. We hypothesize that alterations in the intestinal microbiome are associated with increased immune activation and progression of obesity to NAFLD. The focus of aim 1 is to study the intestinal microbiome of children with NAFLD and non-NAFLD obese controls. High through put 16S rDNA analysis, shotgun sequencing, metagenomic/metatranscriptomic analysis of bacterial genomic DNA/RNA isolated from fecal specimens will be used to identify the characteristic microbiome of each individual and within the 2 different study groups. Pediatric participants with NAFLD will be recruited in collaboration with the existing NIDDK-sponsored multicenter NASH Clinical Research Network (NASH CRN) in collaboration with Dr. Joel Lavine at UCSD. Obese controls will be recruited through the adolescent obesity program at UCSD in collaboration with Dr. Jeffrey Schwimmer. Obese controls will be evaluated for liver steatosis by magnetic resonance spectroscopy (MRS). We will investigate changes in the intestinal microbiome longitudinally in individual participants in response to standard dietary/exercise recommendations, to determine whether the loss of 5% of body weight (accompanied by improvement or resolution of elevated ALT and % hepatic fat) is associated with changes in the microbiome. Aim 2 will characterize the association of systemic inflammation, measured by levels of TNF-1, TGF-21, Lipopolysaccharide binding protein (LBP), and C-Reactive Protein (CRP), with the development of NAFLD, and the intestinal microbiome. Characteristics of the intestinal microbiome indicative of the development of NAFLD will lead to an increased understanding of the disease process. Greater understanding of the role of the intestinal microbiome in the development and progression of NAFLD could lead to the identification of novel biomarkers to predict susceptibility to NAFLD in populations with obesity, and also to novel interventions in the prevention and treatment of this disease through the manipulation and modulation of the intestinal microbiome or its functional metabolic capacity through the use of prebiotics, probiotics, antibiotics, or via other newly revealed mechanisms.